Vaccines are widely used in human and veterinary
medicine as an effective and economic method to control viral and
bacterial diseases. Although generally considered safe, vaccination is
occasionally accompanied by adverse affects. Many adverse affects
related to vaccination are acute and transient, for example, fever,
swelling at the site of the inoculation, and allergic reactions. In
contrast, reports of autoimmune disease following vaccination are
relatively rare. In most instances, it is difficult, if not impossible,
to ascertain that vaccination caused or precipitated the autoimmune
disease. In a recent report, the Advisory Committee on Immunization
Practices in people concluded that there is a causal relation between
diptheria-tetanus-pertussis (DTP) and measles-mumps-rubella (MMR)
vaccination and arthritis, but no evidence of a causal relationship
between these vaccinations and other autoimmune diseases such as
autoimmune hemolytic anemia and Guillain-Barre syndrome (Centers for
Disease Control and Prevention, 1996). Cohen and Shoenfeld (1996) also
stated that the relation between vaccination and autoimmunity is
obscure. They added that there is a need for experimental studies to
address this subject (Cohen and Shoenfeld, 1996).

There has been a growing concern among dog owners and
veterinarians that the high frequency with which dogs are being
vaccinated may lead to autoimmune and other immune-mediated disorders (Dodds,
1988; Smith, 1995). The evidence for this is largely anecdotal and based
on case reports. A recent study observed a statistically significant
temporal relationship between vaccination and subsequent development of
immuno-mediated hemolytic anemia (IMHA) in dogs (Doval and Ciger, 1996).
Although this does not necessarily indicate a causal relationship, it is
the strongest evidence to date for vaccine-induced autoimmune disease in
the dog.

We are investigating the effect of vaccination on dogs
in a series of experimental studies. The goals of these experiments are
(1) to determine if vaccination of dogs affects the function of the
immune system and, in particular, if vaccination results in
autoimmunity; (2) to delineate the mechanisms by which vaccination
results in autoimmunity if this occurs; and (3) to develop alternative
vaccination strategies that will not be accompanied by adverse effects.
The issue that is the focus of this and ongoing studies in our
laboratory is somewhat different from that examined by Duval and Ciger
(1996). In their study, a statistically significant temporal
relationship between the onset of IMHA and prior vaccination suggested
that vaccination caused IMHA or accelerated preexisting IMHA in adult
dogs. Although not documented, it is likely that these middle-aged dogs
had received multiple vaccines prior to the last vaccination. Why this
last vaccination suddenly triggered the onset of IMHA is unknown. In
contrast, our studies examine if vaccination of dogs at a young
age causes alterations in the immune system, including the production of
autoantibodies, that could eventually lead to autoimmune disease in
susceptible individuals. In this paper, we report on the findings of the
first study in which a group of vaccinated dogs and a group of
unvaccinated dogs were followed for 14 weeks after the first
vaccination.

II. Materials and Methods

A. Animals

Two pregnant Beagle dogs were purchased from a commercial breeder.
The animals whelped in the Animal Facility of the Purdue University
School of Veterinary Medicine and the pups were weaned at 6 weeks of
age. Five pups were assigned to one of two groups, a vaccinated and an
unvaccinated group, based on body weight, gender, and litter of origin.
The vaccinated and unvaccinated group of dogs were housed in separate
rooms.

The dogs were examined daily. Rectal temperature and body weight were
recorded twice a week. Blood samples were collected from the jugular
vein prior to each vaccination and 2, 5, 7, and 14 days following
vaccination for hematology, endocrinology, and viral serology. Blood
samples collected on days 5 and 14 following vaccination were also used
for lymphocyte phenotyping and lymphocyte proliferation assays, and
blood samples collect at 7 days following vaccination were used for the
detection of autoantibodies.

B. Vaccination Schedule

The dogs in the vaccinated group were injected subcutaneously with a
commercially available multivalent vaccine, Vanguard-5 CV/L (Pfizer,
Croton, CT) at 8, 10, 12, 16, and 20 weeks of age according to the
instructions of the manufacturer. They were injected subcutaneously with
an inactivated rabies vaccine, Imrab-2 (Rhone-Mericux, GA) at 16 weeks
of age. The unvaccinated group of dogs received subcutaneous injections
of sterile saline at the same time points.

Both groups of dogs were injected subcutaneously with 1 mg of keyhole
limpet hemocyanin (KLH, Calbiochem) in RIBI-adjuvant at week 20.

C. Viral Serology

Serum samples collected at 6 weeks of age and 0, 2, 5, 7, and 14 days
after each vaccination were assayed for the presence of antibodies to
canine distemper virus by serum neutralization test, and for antibodies
against canine parvovirus by hemagglutination inhibition test. Serum
samples were analyzed for antibodies against rabies virus at 16 and 20
weeks of age by a rapid fluorescent focus inhibition test.

D. Hematology

Blood samples were collected at 0, 2, 5, 7, and 14 days after each
vaccination for hematocrit, corrected white blood cell count and
differential, and platelet counts.

E. Endocrinology

Plasma and serum samples collected at 0, 2, 5, 7, and 14 days after
each vaccination were assayed for curtisol, triiodothymonine (T3), and
thyroxine (T4) by radioimmunoassay.

F. Immunology

Lymphocyte phenotyping was used. Whole blood was stained with a panel
of mouse monoclonal antibodies, followed by F(ab')2 goat anti-mouse IgG
(Jackson Research Laboratories). The monoclonal antibodies used were
CA2.1D6 (anti-CD21), CA15.8G7 (anti-TCRoB), CA20.8H1 (anti-TCRv81,
12.125 (anti-CD4), and 1.140 (anti-CD8). The characteristics of these
monoclonal antibodies have been described (Gebhard and Carter, 1992;
Moore et al., 1995). Following red blood cell lysis and fixation in 2%
paraformaldehyde, the cells were analyzed by flow cytometry.

G. Lymphocyte Blastogenosis Assay

Heparinized blood samples were diluted 1:10 in RPM1-1640 and
distributed in the wells of a 96-well plate. Triplicate samples were
incubated for 96 hours in the presence of medium only, 2.5 and 5 pg/ml
PHA, 5 and 10 pg/ml Concenavalin A (Con A) and 1 and 10 pg/ml PWM.
During the last 24 hours of incubation the wells were pubed with 0.5 uCi
of H-thymidine. The cells were harvested with a 96-well cell harvester,
and the incorporation of radioactivity was measured in a TopCount
scintillation counter (Packard Instrument Co., Meriden, CT).

H. Enzyme-Linked Immunosorbent Assay (ELISA)

The presence of antibodies reactive with homologous and heterologous
antigens in serum samples collected at 22 weeks of age was analyzed by
an indirect ELISA. High-binding ELISA plates (Costar, Cambridge, MA)
were coated with 10 pg/ml of antigen in 0.1 M bicarbonate buffer. The
wells were rinsed and incubated for 1 hour with phosphate-buffered
saline (PBS)/0.1% Tween. Serum samples were diluted 1:10 in PBS and
added to the wells in triplicate. Following incubation, the wells were
rinsed and incubated with alkaline phosphatase labeled goat anti-dog IgG
(Kirkegnard and Perry, Gaithersburg, MD). Alkaline phosphatase activity
was measured after addition of p-NPP substrate at 405 nm in a microplate
reader (Molecular Devices, Menlo Park, CA).

Essentially the same procedure was used to measure the presence of
antibodies against KLH. Alkaline phosphatase labeled anti-dog IgM and
IgG were used as secondary reagents.

I. Necropsy

At 22 weeks of age, the dogs were killed by intravenous injection of
barbiturates, and a complete necropsy performed. Tissue samples were
collected in 10% buffered formalin and processed for light microscopic
examination. The tissues that were examined included the spleen, lymph
nodes, tonsils, thymus, Psyer's patches, adrenal glands, thyroid glands,
pituitary gland, pancreas, heart, lung, kidney, liver, and brain.

J. Statistical Analysis

Data were analyzed for significant differences between groups by
Student's t test or repeated measures ANOVA and a significant change
over time using a repeated measures ANOVA.

III. Results

A. Viral Serology

None of the pups had detectable antibodies against canine distemper
virus and canine parvovirus at 6 weeks of age and against rabies virus
at 16 weeks of age. The unvaccinated dogs remained seronegative for
these three viruses during the course of the study. The dogs that were
immunized developed titers against CDV (maximum titers ranged from 1:48
to 1:1024), CPV-2 (1:320 to 1:1280), and rabies (1:25 to 1:1000).

B. Clinical Observations, Hematology, and
Endocrinology

No differences between the unvaccinated and vaccinated groups were
found for rectal temperature, body weight, and hematologic values.

There were no significant differences between unvaccinated and
vaccinated dogs for concentrations of cortisol, T3, and T4. However, a
significant (p<0.02) change was observed over time for each of these
three hormones. The plasma concentration of cortisol decreased from a
mean of 41.1 ng/ml at 8 weeks of age to 17.6 ng/ml at 22 weeks of age.
The concentration of T4 also decreased, from 31.1 ng/ml at 8 weeks of
age to 22.8 ng/ml at 22 weeks of age. The concentration of T3 increased
from 0.63 ng/ml at 8 weeks of age to 1.1 ng/ml at 22 weeks of age.

C. Immunology

No differences were observed unvaccinated and vaccinated dogs for
lymphocyte subpopulations or for the proliferative response to any of
the mitogens tested.

The response of both groups of dogs to KLH was similar. There was no
statistically significant difference in the KLH-specific IgM and IgG
concentrations in the serum (not shown).

At 8 weeks of age, antibodies against homologous and conserved
heterologous antigens were negligible in the serum of the dogs. At 22
weeks of age there was a significant increase of IgG antibodies reactive
with 10 of 17 antigens in the vaccinated dogs versus no increase in the
unvaccinated dogs (Table I). The increase of optical density was modest
for 8 of these 10 antigens, but a large increase was observed for
fibronectin and laminin. All vaccinated dogs developed high levels of
fibronectin-specific IgG antibodies. Similar levels of IgG anti-fibronectin
antibodies were observed when bovine fibronectin was substituted by
human or mouse fibronectin (not shown). The concentration of anti-fibronectin
antibodies began to increase after the second vaccination in three dogs
and after the third vaccination in the other two vaccinated dogs, and
reached a maximum level after the fourth vaccination (Fig. 1). To
determine if the antibodies had a preferential reactivity with a
particular part of the bironectin molecule, we tested the reactivity of
serum samples with two fragments of the fibronectin. The 30-kDa
fragments contains the heparin-binding domain of fibronectin, whereas
the 45-kDa fragment contains the collagen-binding domain. As shown in
Fig. 2, little reactivity was observed with the 45-kDa fragment, but
significant reactivity was observed with the 30-kDa fragment.

High levels of anti-laminin antibodies were observed in the serum of
three of the five vaccinated dogs at 22 weeks of age. One dog had high
levels at 17 weeks of age, whereas the other two dogs did not devlop
high levels until the end of the study.

High levels of antibodies reactive with skeletal muscle myosin and
myoglobin were observed in both groups of dogs at 22 weeks of age. The
antibody levels increased at 11 weeks of age in three dogs, at 13 weeks
of age in another three dogs, and at 17 weeks of age in the remaining
four dogs.

D. Necropsy

Gross and light microscopic examination of the tissues of the dogs
revealed no significant lesions. The thyroid gland of one of the
vaccinated dogs had a small lymphoid nodule with obliteration of
adjacent thyroid follicles.

IV. Discussion

In this study, we exhaustively evaluated the effects
of vaccination with a multivalent vaccine and a rabies vaccine on the
immune system of young dogs. Vaccination did not cause immunosuppression
or alter the response to an unrelated antigen (KLH). In contrast to an
earlier study (Mastro et al., 1986), but in agreement with other work
(Phillips and Schultz, 1987), we did not observe a transient lymphopenia
in the dogs at any time. However, vaccination did induce autoantibodies
and antibodies to conserved heterologous antigens. The pathogenic
significance of these autoantibodies is presently uncertain. We did not
find any evidence of autoimmune disease in the vaccinated dogs, but the
study was terminated when the dogs were 22 weeks of age, well before
autoimmune diseases usually become clinically apparent. It is likely
that genetic and environmental factors will trigger the onset of
clinical autoimmune disease in a small percentage of the animals that
develop autoantibodies. For practical and economic reasons, only a small
number of dogs can be followed in an experimental study, and clinical
autoimmune disease may, therefore, never be observed. The principal
value of an experimental study is that it enables us to determine the
frequency of autoantibody responses and the mechanism(s) that cause
vaccines to induce autoantibodies.

We used two vaccines, a multivalent vaccine and an inactivated rabies
vaccine of a particular commonly used brand. We consider it unlikely
that the observed autoantibodies were specifically induced in response
to those brands of vaccine and this phenomenon will likely occur with
other commercial vaccines. In a follow-up study, we have observed
similar autoimmune phenomena in dogs immunized with the multivalent
vaccine only and in dogs immunized with the rabies vaccine only
(unpublished observations).

There was a marked increase of autoantibodies to the skeletal muscle
proteins, myoglobin and myosin, in both groups of dogs. The reason for
the appearance of these antibodies is uncertain, but it may be the
result of the frequent blood sampling of the dogs. The dogs were bled
five times following each vaccination, and some tissue trauma was
unavoidable.

We examined the thyroid and adrenal cortical function in the dogs,
and did not find evidence of any abnormality. Autoimmune thyroiditis is
one of the most common autoimmune diseases of dogs, and it has been
suggested that the apparent increase of this condition in dogs is
related to the increased frequency of vaccination with modified live
vaccines. There was no increase of anti-thyroglobulin antibodies in the
vaccinated animals, or other evidence of thyroid dysfunction. However,
the lymphoid nodule found in the thyroid gland of one of the vaccinated
dogs may be an early manifestation of thyroiditis, a common lesion in
purpose bred Beagles (Fritz et al., 1970).

The most strikingly increased concentrations of autoantibodies were
directed against fibronectin and laminin. Fibronectin is widely
distributed in the body as a component of the extracellular matrix and
plasma. The anti-fibronectin antibodies were reactive with fibronectin
of bovine, murine, and human origin. Although we have not yet
demonstrated that they also react with canine fibronectin, this is very
likely, since fibronectin is highly conserved between species. Anti-fibronectin
antibodies have been found in human patients with systemic lupus
erythematosus (SLE) and rheumatoid arthritis, and a patient with a
poorly defined connective tissue disease (Henane et al., 1986; Atta et
al., 1994, 1995; Girard et al., 1995). The anti-fibronectin antibodies
in four human SLE patients were directed against the collagen-binding
domain (Atta et al., 1994), in contrast to the anti-fibronectin
antibodies in the vaccinated dogs, which showed no affinity for this
domain. The anti-fibronectin antibodies in the human patient with
connective tissue disease showed reactivity with the cell-binding domain
of fibronectin (Girard et al., 1995).

Anti-fibronectin antibodies have been experimentally induced in
rabbits by immunization with human fibronectin in complete Freund's
adjuvant (Murphy-Ullrich et al., 1984). The antibodies were reactive
with both human and rabbit fibronectin. The rabbits subsequently
developed a glomernlopathy with granular deposits suggestive of immune
complexes in the glomcrular basement membrane. Anti-fibronectin
antibodies have been induced in mice by multiple injections of
homologous fibronectin without adjuvant (Murphy-Ulrich et al., 1986).
The titer of anti-fibronectin antibodies was much lower in mice
immunized with native fibronectin than in mice immunized with de-natured
fibronectin. However, in both groups, immune complexes were present in
the serum and in the glomerali (Murphy-Ullrich et al., 1986). Light
microscopic examination of the glomerali of the kidneys of vaccinated
dogs did not reveal evidence of glomerunfpathy, but we cannot exclude
the possibility of sub-light microscopic lesions.

Anti-laminin antibodies were prevalent in the serum of three of the
five vaccinated dogs. Anti-laminin antibodies are increased in human
patients with SLE, rheumatoid arthritis, and vasculitis. Injection of
polyclonal anti-laminin antibodies into rats resulted in
glocnerulopathay and proteinuria (Abrahamson and Caulfield, 1982)>
Anti-laminin antibodies have also been implicated in glomaerular disease
in rats induced by mercuric chloride (Aten et al., 1995).

The mechanisms that may underlie the production of autoantibodies
following vaccination are unknown, but at least four mechanisms can be
proposed: cross-reactivity with vaccine-components, somatic mutation of
immunoglobulin variable genes, "bystander activation" of
self-reactive lymphocytes, and polyclonal activation of lymphocytes.
Perhaps the simplest and most likely mechanism is that of
cross-reactivity of vaccine and self-antigens. (Schattner and
Rager-Ziaman, 1990), the most likely sources of cross-reactive epitopes
are bovine serum and cell culture components. These are present in
almost all vaccines as residual components of the cell culture necessary
to generate vaccine viruses and may purposely be added to the vaccine as
a stabilizer. In the presence of an adjuvant, these bovine products
stimulate a strong immune response and induce antibodies that
cross-react with conserved canine antigens. Thus, the strong response to
fibronectin in the vaccinated dogs is most likely the result of the
injection of bovine fibronectin contaminants in the vaccine. Indeed,
this is essentially identical to the protocol used to produce anti-fibronectin
antibodies in rabbits with human fibronectin in complete Freund's
adjuvant (Murphy-Ullrich et al., 1984), as mentioned above. The lower
response to other antigens (e.g., cardiolipin and laminin) may be due to
a lower concentration of these antigens in the vaccine or lower
immunogenicity.

During every immune response, self-reactive B and T lymphocytes are
generated and activated. This is the result of somatic mutation and
bystander activation. Under normal conditions, this will not lead to
significant production of autoantibodies, because of the selection
process in the germinal centers of lymph nodes. In the germinal centers
only B cells that successfully compete for interaction with antigen
presented on the surface of follicular dendritic celia will be allowed
to survive (MacLennan, 1994). These B cells generally have high-affinity
receptors for the antigen to which the immune response was induced. B
cells with low affinity for the antigen or affinity for other antigens,
including self-antigens, will undergo programmed cell death. The B cells
with high-affinity receptors express bc1-2, which may rescue them from
programmed cell death (MacLennan, 1994). This mechanism was elegantly
demonstrated in mice immunized with a nominal antigen phosphorylcholine
(Ray et al., 1996). A single point mutation in the hypervariable region
of the expressed immunoglobulin genes was sufficient for the
phosphorylcholine-specific B cells to acquire specificity for DNA.
However, it was only possible to demonstrate DNA-specific B cells by
fusing germinal center B cells with celia that expressed high levels of
bc1-2, thereby rescuing them from programmed cell death (Ray et al.,
1996). An increased expression of bc1-2 was observed in thymic lymphoid
follicles of patients with myasthenia gravis, suggesting that failure to
delete self-reactive B cells in these patients may lead to autoimmune
disease (Shiono et al., 1997). While this may seem an attractive
hypothesis to explain autoimmune phenomena in human beings and dogs,
there is currently no evidence that this is a common mechanism.

Finally, polyclonal activation of lymphocytes, including activation
of self-reactive lymphocytes, is a possible mechanism of vaccine-induced
autoimmunity. Certain viruses and bacteria have superantigen or mitegen
activity (Schwarts, 1993). This could also be the case for the microbial
products included in the vaccines. The present study does not support
this mechanism. Firstly, antibodies were observed against 10 of 17
antigens tested. Secondly, the anti-fibronectin antibodies did not react
with any portion of the fibronectin molecule, but instead, reacted most
strongly with the heparin binding domain. These observations indicate
that the appearance of autoantibodies in the serum of vaccinated dogs is
an antigen-driven process and not caused by polyclonal activation. As
argued earlier, the main antigens implicated are cell culture
contaminants and bovine serum components.

In the dog, certain autoimmune diseases occur more frequently in
particular breeds of dogs, indicating genetically determined
susceptibility (Dodds, 1983; Happ, 1995). There is abundant evidence
from studies in rodents and human beings that the magnitude of the
antibody response and the susceptibility to autoimmune disease are in
part genetically determined (Schwartz, 1993). It is likely that genetic
factors also determine the susceptibility to vaccine-induced
autoimmunity. That this is indeed the case is suggested by the finding
that only three of the five vaccinated dogs developed a strong anti-laminin
antibody response and that the kinetics of the anti-fibronectin response
differed between individual animals. Identification of susceptibility
genes will be important, because it may shed light on the pathogenesis
of the autoimmunity. In addition, it will provide genetic tests that
will enable dog breeders to monitor the susceptibility of their breeding
stock to vaccine-induced autoimmunity.

Although the pathogenic significance of the vaccine-induced
autoantibodies is still unclear, there are a number of ways to prevent
their induction. Not vaccinating dogs is not a viable option, because
the benefits of vaccination clearly outweigh the still uncertain risks
of immune-mediated disease. However, since bovine serum components in
the vaccine may be responsible for the majority of autoantibodies,
elimination of these bovine components may avoid this problem. This
could be accomplished by substituting homologous serum for bovine serum.
However, as mentioned earlier, anti-fibronectin antibodies may still be
induced by immunization with homologous fibronectin. New generations of
vaccines, especially naked DNA vaccines, are free of serum components,
and these should not induce autoantibodies. A recent study in mice
indicates that DNA vaccination does not induce or accelerate autoimmune
disease (Mer et al., 1997). Finally, mucosal vaccines are less likely to
induce autoantibodies than parenterally administered vaccines. Depending
on the formulation of the vaccine, soluble serum components are less
likely to be absorbed via the mocosal surface, and, in fact, may induce
tolerance instead of autoantibodies (Weiner et al., 1994).

In conclusion, we have demonstrated that vaccination of dogs using a
routine protocol and commonly used vaccines, induces autoantibodies. The
autoantibody response appears to be antigen driven, probably directed
against bovine antigens that contaminate vaccines as a result of the
cell culture process and/or as stabilizers. The pathogenic significance
of these autoantibodies has not yet been determined.

Acknowledgments

The authors thank Cheryl Anderson and Julie Tobelski-Crippen for
animal care and technical support, and Nita Glickman for data
management. This work is supported by the John and Winifred Hayward
Foundation.